Interferons (IFNs) play a critical role in innate defense against infection with RNA and DNA viruses. A detailed understanding of how these critical cytokines inhibit viral infection might lead to pharmacologic approaches to induce resistance to multiple potentially dangerous agents without the necessity of inducing prior antigen-specific immunity. Given the large number of potential biological weapons for which no vaccine exists, this is an important goal. IFNs work via induction or repression of proteins that interfere with viral replication or alter the function of immune cells. In this grant we will focus on IFN regulated genes that inhibit viral replication. There are hundreds of IFN regulated genes, the function of only a few of which is understood in detail. Sorting through the many IFN regulated genes to find those with specific antiviral functions is a daunting task. Our preliminary data, and work from others, suggests that critically important IFN inducible antiviral molecules remain unidentified. We have succeeded in developing methods for discovering these important antiviral molecules. Based on these considerations, we propose to identify and analyze the mechanisms of action of novel IFN induced antiviral proteins. We will use two screens for antiviral proteins: (i) expression cloning of proteins that block viral replication, and (ii) use of gene chip analysis coupled to a Sindbis vector based complementation assay to identify proteins with antiviral function in vivo. We have successfully used this second approach to identify an IFN inducible antiviral protein, the ubiquitin-like protein ISG15. We have begun to analyze the role of ISG15 in resistance to several viruses including viruses in Class A and B. In this proposal we will analyze ISG 15 action in infected cells in detail (Aim 1), and continue to use the screens we have set up to identify additional novel IFN induced antiviral molecules (Aim 2). Aim 1. Determine the molecular mechanism(s) of the antiviral effect of ISG15. Aim 2. Identify candidate novel antiviral proteins and prove their physiologic relevance.